Bacteria Detection and Differentiation Using Impedance Flow Cytometry.
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ABSTRACT:
SUBMITTER: Clausen CH
PROVIDER: S-EPMC6210208 | biostudies-literature | 2018 Oct
REPOSITORIES: biostudies-literature
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Project description:Impedance flow cytometry (IFC) is a versatile lab-on-chip technology which enables fast and label-free analysis of pollen grains in various plant species, promising new research possibilities in agriculture and plant breeding. Hazelnut is a monoecious, anemophilous species, exhibiting sporophytic self-incompatibility. Its pollen is dispersed by wind in midwinter when temperatures are still low and relative humidity is usually high. Previous research found that hazelnut can be characterized by high degrees of pollen sterility following a reciprocal chromosome translocation occurring in some cultivated genotypes. In this study, IFC was used for the first time to characterize hazelnut pollen biology. IFC was validated via dye exclusion in microscopy and employed to (i) follow pollen hydration over time to define the best pre-hydration treatment for pollen viability evaluation; (ii) test hazelnut pollen viability and sterility on 33 cultivars grown in a collection field located in central Italy, and two wild hazelnuts. The accessions were also characterized by their amount and distribution of catkins in the tree canopy. Pollen sterility rate greatly varied among hazelnut accessions, with one main group of highly sterile cultivars and a second group, comprising wild genotypes and the remaining cultivars, producing good quality pollen. The results support the hypothesis of recurring reciprocal translocation events in Corylus avellana cultivars, leading to the observed gametic semi-sterility. The measured hazelnut pollen viability was also strongly influenced by pollen hydration (R adj2 = 0.83, P ≤ 0.0001) and reached its maximum at around 6 h of pre-hydration in humid chambers. Viable and dead pollen were best discriminated at around the same time of pollen pre-hydration, suggesting that high humidity levels are required for hazelnut pollen to maintain its functionality. Altogether, our results detail the value of impedance flow cytometry for high throughput phenotyping of hazelnut pollen. Further research is required to clarify the causes of pollen sterility in hazelnut, to confirm the role of reciprocal chromosome translocations and to investigate its effects on plant productivity.
| S-EPMC7753158 | biostudies-literature
Project description:The development of reliable and cost-efficient methods to assess the toxicity of nanomaterials (NMs) is critical for the proper identification of their impact on human health and for ensuring a safe progress of nanotechnology. In this study, we investigated the reliability and applicability of label-free impedance flow cytometry (IFC) for in vitro nanotoxicity screening, which avoids time-consuming labelling steps and minimizes possible NM-induced interferences. U937 human lymphoma cells were exposed for 24?h to eight different nanomaterials at five concentrations (2, 10, 20, 50, and 100??g/mL). The NMs' effect on viability was measured using IFC and the results were compared to those obtained by trypan blue (TB) dye exclusion and conventional flow cytometry (FC). To discriminate viable from necrotic cells, the IFC measurement settings regarding signal trigger level and frequency, as well as the buffer composition, were optimised. A clear discrimination between viable and necrotic cells was obtained at 6?MHz in a sucrose-based measurement buffer. Nanomaterial-induced interferences were not detected for IFC. The IFC and TB assay results were in accordance for all NMs. The IFC was found to be robust, reliable and less prone to interferences due to the advantage of being label-free.
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Project description:IntroductionAn efficient and reliable method to estimate plant cell viability, especially of pollen, is important for plant breeding research and plant production processes. Pollen quality is determined by classical methods, like staining techniques or in vitro pollen germination, each having disadvantages with respect to reliability, analysis speed, and species dependency. Analysing single cells based on their dielectric properties by impedance flow cytometry (IFC) has developed into a common method for cellular characterisation in microbiology and medicine during the last decade. The aim of this study is to demonstrate the potential of IFC in plant cell analysis with the focus on pollen.MethodDeveloping and mature pollen grains were analysed during their passage through a microfluidic chip to which radio frequencies of 0.5 to 12 MHz were applied. The acquired data provided information about the developmental stage, viability, and germination capacity. The biological relevance of the acquired IFC data was confirmed by classical staining methods, inactivation controls, as well as pollen germination assays.ResultsDifferent stages of developing pollen, dead, viable and germinating pollen populations could be detected and quantified by IFC. Pollen viability analysis by classical FDA staining showed a high correlation with IFC data. In parallel, pollen with active germination potential could be discriminated from the dead and the viable but non-germinating population.ConclusionThe presented data demonstrate that IFC is an efficient, label-free, reliable and non-destructive technique to analyse pollen quality in a species-independent manner.
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Project description:ObjectivesConventional microbiological procedures for the isolation of bacteria from biological fluids consist of culture on solid media and enrichment broth. However, these methods can delay the microbiological identification for up to 4 days. The aim of this study was to evaluate the analytical performance of Sysmex UF500i (Sysmex, Kobe, Japan) as a screening method for the detection of bacteria in different biological fluids in comparison with direct Gram staining and the conventional culture on solid media and enrichment broth.MethodsA total of 479 biological fluid samples were included in the study (180 ascitic, 131 amniotic, 56 synovial, 40 cerebrospinal, 36 pleural, 24 peritoneal, 9 bile and 3 pericardial fluids). All samples were processed by conventional culture methods and analyzed by flow cytometry. Direct Gram staining was performed in 339 samples. The amount of growth on culture was recorded for positive samples.ResultsBacterial and white blood cell count by flow cytometry was significantly higher among culture positive samples and samples with a positive direct Gram stain compared to culture negative samples. Bacterial count directly correlated with the amount of growth on culture (Kruskall-Wallis H χ2(3) = 11.577, p = 0.009). The best specificity (95%) for bacterial count to predict culture positivity was achieved applying a cut-off value of 240 bacteria/μL.ConclusionsBacterial and white blood cell counts obtained with flow cytometry correlate with culture results in biological fluids. Bacterial count can be used as a complementary method along with the direct Gram stain to promptly detect positive samples and perform other diagnostic techniques in order to accelerate the bacterial detection and identification.
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Project description:Directed localization of kinases within cells is important for their activation and involvement in signal transduction. Detection of these events has been largely carried out based on imaging of a low number of cells and subcellular fractionation/Western blotting. These conventional techniques either lack the high throughput desired for probing an entire cell population or provide only the average behaviors of cell populations without information from single cells. Here we demonstrate a new tool, referred to as microfluidic electroporative flow cytometry, to detect the translocation of an EGFP-tagged tyrosine kinase, Syk, to the plasma membrane in B cells at the level of the cell population. We combine electroporation with flow cytometry and observe the release of intracellular kinase out of the cells during electroporation. We found that the release of the kinase was strongly influenced by its subcellular localization. Cells stimulated through the antigen receptor have a fraction of the kinase at the plasma membrane and retain more kinase after electroporation than do cells without stimulation and translocation. We are able to differentiate a cell population with translocation from one without it with the information collected from individual cells of the entire population. This technique potentially allows detection of protein translocation at the single-cell level. Due to the frequent involvement of kinase translocations in disease processes such as oncogenesis, our approach will have utility for kinase-related drug discovery and tumor diagnosis and staging.
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